SRP-27 (sarcoplasmic reticulum protein of 27 kDa) is a newly identified integral membrane protein constituent of the skeletal muscle SR (sarcoplasmic reticulum). We identified its primary structure from cDNA clones isolated from a mouse skeletal muscle cDNA library. ESTs (expressed sequence tags) of SRP-27 were found mainly in cDNA libraries from excitable tissues of mouse. Western blot analysis confirmed the expression of SRP-27 in skeletal muscle and, to a lower extent, in heart and brain. Mild trypsin proteolysis combined with primary-structure prediction analysis suggested that SRP-27 has four transmembrane-spanning alpha helices and its C-terminal domain faces the cytoplasmic side of the endo(sarco)plasmic reticulum. The expression of SRP-27 is higher in fast twitch skeletal muscles compared to slow twitch muscles and peaks during the first month of post-natal development. High-resolution immunohistochemistry and Western blot analysis of subcellular fractions indicated that SRP-27 is distributed in both longitudinal tubules and terminal cisternae of the SR, as well as in the perinuclear membrane systems and the nuclear envelope of myotubes and adult fibres. SRP-27 co-sediments with the RyR (ryanodine receptor) macromolecular complex in high-salt sucrose-gradient centrifugation, and is pulled-down by anti-RyR as well as by maurocalcin, a well characterized RyR modulator. Our results indicate that SRP-27 is part of a SR supramolecular complex, suggesting the involvement of SRP-27 in the structural organization or function of the molecular machinery underlying excitation–contraction coupling.
The skeletal muscle SR (sarcoplasmic reticulum) is an intracellular membrane compartment that controls the myoplasmic calcium concentration and therefore plays a crucial role in E–C (excitation–contraction) coupling [1–3]. E–C coupling occurs in the triad, an intracellular junction formed by the association of the transverse tubules and the SR terminal cisternae [2–4]. E–C coupling is initiated by conformational changes of the L-type voltage-dependent Ca2+ channel DHPR (dihydropyridine receptor), the voltage sensor localized within the membrane of the T tubular system [4,5]. Activation of Ca2+ release from the SR terminal cisternae is due to the direct transmission of conformational changes of DHPRs to RyRs (ryanodine receptors). This model implies a close association between the SR terminal cisternae and the T tubular membrane, and as well as the two channels DHPR and RyR [5–10]. Detailed analysis of highly purified triad membrane fractions revealed that their protein composition is quite complex. The known protein components are: RyR, the SR Ca2+ release channel; DHPR; triadin; HRCP, the histidine rich Ca2+ binding protein; the 90 kDa JFP (junctional face membrane protein); calsequestrin; mitsugumins; junctophilin; JP45; junctate; the 32 kDa ADT/ATP carrier [11–21] and a number of other polypeptides corresponding to still-unidentified proteins. Because of their localization in the junctional SR membrane, these proteins probably play a role either in the organization of the triad during development or in the mechanism of signal transduction in skeletal muscle. In this study, we discovered a 27 kDa protein that we named SRP-27 (for sarcoplasmic reticulum protein-27 kDa). We show that this polypeptide is present in skeletal muscle SR membranes as well as in the nuclear/endo(sarco)plasmic reticulum membrane of excitable tissues including heart and brain. SRP-27 co-sediments with the RyR complex, and its level of expression depends on fibre type differentiation and on the development stage.
Protein G–Sepharose, PCR primers, peroxidase-conjugated Protein A and Protein A–Sepharose were from Sigma/Fluka; the pGEX-5X-3 plasmid, [35S]-methionine and Protein A–Sepharose were from GE Healthcare. The pEYFP-N1 [where EYFP is enhanced YFP (yellow fluorescent protein)] plasmid was from Clontech. Nitrocellulose membrane was from Schleicher & Schuell BioScience. Protein-molecular-mass markers were from Bio-Rad. All other chemicals were reagent or highest available grade. Rats (Wistar) were bred in-house. Rabbits (New Zealand White) were purchased from Charles Rivers Laboratories (Calco, Italy). All procedures were performed in accordance with the stipulations of the Helsinki Declarations for care and use of laboratory animals.
SRP-27 cDNA cloning
Total RNA was isolated from rat limb skeletal muscles using Tri-Reagent (Molecular Research Center) following the instructions provided by the manufacturer. Total RNA was converted into cDNA using the cDNA Synthesis Kit from Roche Applied Science using the T17-adapter primer 5′-GACTCGAGTCGACATCGAT-(17)-3′ in order to reverse-transcribe mRNA and obtain cDNA with an adapted sequence in the 3′-end compatible to perform 3′-RACE-PCR (where RACE is rapid amplification of cDNA ends). All clones were sequenced (Microsynth AG, Balgach, Switzerland). The ORF (open reading frame) cDNA of rat SRP-27 digested with EcoRI and XhoI was used as a probe to screen a mouse skeletal muscle UNI-ZAP XR cDNA library as previously described . A clone of 2123 bp containing the entire ORF was pulled out and sequenced (Microsynth AG, Balgach, Switzerland).
Cryosections of mouse diaphragm muscle were immunostained as described in Flucher et al. . Primary antibodies and working dilutions were: affinity-purified rabbit anti-SRP-27 (see below; 1:100); affinity-purified rabbit anti-RyR1 (1:2000) ; mouse monoclonal anti-panRyR (C-34, Alexis, Lausen, Switzerland; 1:1000); mouse monoclonal anti-SERCA2a (where SERCA is sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) (MA3-911; Affinity Bio Reagents, Neshanic Station, NJ, U.S.A.; 1:1000); mouse monoclonal antibody against α-actinin (1:1000). Secondary antibodies Alexa488- and Alexa595-conjugated goat-anti-rabbit and goat-anti-mouse IgG (Molecular Probes) were used at dilutions of 1:4000. Images were recorded on a Zeiss Axiophot microscope with a cooled CCD camera and METAVUE image-processing software (Universal Imaging, West Chester, PA, U.S.A.).
Western blot staining
SDS/PAGE electrophoresis and indirect immunoenzymatic staining were carried out as previously described . Protein concentration was determined with the DC Protein Assay kit (Bio-Rad) following the instructions provided by the manufacturer and using bovine serum albumin as standard.
Polyclonal antibody production
Details are described in the Supplementary Figure 1 at http://www.BiochemJ.org/bj/411/bj4110343add.htm.
Subcellular fraction preparation
Expression in COS-7 and TSA cells
COS-7 African green monkey kidney cells were transfected with the SRP-27-pEYFP-N1 plasmid or the pEYFP-N1 plasmid as previously described . Transfected COS-7 cells were imaged 48 h after transfection with a Nikon Eclipse TE 2000-U inverted fluorescent microscope equipped with a 100× Plan-Apo oil immersion objective and using a D1 digital camera (Nikon). TSA (human embryonic kidney cell, SV40 [(simian virus 40) transformed] cells were transfected with the SRP-27-pEYFP-N1 plasmid or the pEYFP-N1 plasmid as control using calcium phosphate as previously described . Cells were used 48 h after transfection and the total microsomal fraction was prepared as described .
Protein (15 μg) was digested with increasing concentrations of trypsin for 2 min at room temperature (20 °C). The reaction was blocked by adding soybean trypsin inhibitor and samples were analysed by staining the Western blot with anti-GFP (green fluorescent protein) BD-Living Colors A.v. Peptide (Clontech) or anti-calreticulin (Santa Cruz).
Vesicles derived from the rat skeletal muscle membrane heavy (R4) fractions were solubilized in the presence of 1 or 0.2 M NaCl, 1% CHAPS, 0.003% egg phosphatidylcholine (L-α-Lecithin, egg yolk), 10 mM Hepes (pH 7.4), 1 mM DTT (dithiothreitol) and protease inhibitors for 30 min at 4 °C. Unsolubilized proteins were removed by centrifugation and soluble proteins were separated on a 5–20% sucrose gradient by centrifugation at 25000 rev./min for 16 h at 4 °C in a SW40 Ti Beckman rotor . Fractions were collected and analysed by SDS/PAGE and immunoblotted with anti-SRP27.
Pull-down and co-immunoprecipitation experiments
Proteins solubilized from the heavy SR R4 fractions (see above) were incubated with streptavidin polystyrene beads (Dynabeads M-280 Streptavidin, Dynal-Biotech) coated with biotinylated maurocalcine (40 mM) in PBS in the presence of protease inhibitors and BSA (0.1 mg/ml). After incubation, the beads were collected, washed and the bound proteins were analysed either by immunoblot or autoradiography.
For co-immunoprecipitation experiments, 1 mg of rat skeletal muscle heavy SR was solubilized (4 °C, 30 min) in a buffer containing 1% CHAPS, 0.2 M NaCl, 10 mM Hepes (pH 7.4), 0.003% lipids (L-α-Lecithin, egg yolk), 1 mM DTT and protease inhibitor cocktail in a final volume of 1 ml. After centrifugation (30 min at 100000 g, 4 °C), solubilized proteins were diluted 10 times with 0.2 M NaCl, 10 mM Hepes (pH 7.4) and protease inhibitor cocktail and incubated for 2 h at room temperature (20 °C) with Protein A–Sepharose beads coated with anti-SRP-27 (50 μg) or Protein G–Sepharose beads coated with anti-RyR (10 μg). Beads were then washed three times with PBS and the bound proteins were eluted with cracking buffer (6 M urea, 10 mM sodium phosphate, pH 7.2, 1% SDS, 1% 2-mercaptoethanol). Proteins present in the void, last wash and bound to the beads were separated by SDS/PAGE and blotted on to nitrocellulose.
In vitro transcription
In vitro transcription  was performed using the TnT quick coupled transcription/translation system (Promega) according to the manufacturer's instructions.
Statistical analysis and software
Statistical analysis was performed using the Student's t test for unpaired samples; means were considered statistically significant when the P value was <0.05. Images were processed using Gimp 2.2.13 and Corel Photopaint 6.0 software. Blast alignments were performed on the NCBI web site using BLAST 2.2.8 release or higher. Multiple sequence alignments were performed using the Clustal W algorithm available from the Swiss node of the European Molecular Biology Network. Transmembrane domain prediction was obtained using TMhMM 2.0 on the Biology Workbench web site [25,26].
RESULTS AND DISCUSSION
The biochemical and molecular characterization of the proteins present in SR membrane is an important task for understanding the mechanisms underlying Ca2+ homoeostasis in skeletal muscle under normal and pathological conditions. To identify novel proteins of the skeletal muscle SR involved in E–C coupling, we performed MS analysis of the protein constituents of the junctional face membrane. Proteins of the junctional face membrane were separated on SDS/PAGE, and the bands corresponding to polypeptides having a molecular mass ranging between 10 and 100 kDa were processed for electrospray MS analysis. This approach allowed us to identify a 19-amino-acid long peptide sequence from a band migrating as a single polypeptide chain of 27 kDa (Figure 1, Rabbit pep.). Its primary sequence matched that of a hypothetical protein in the NCBI database. We named this novel SR protein SRP-27, for SR protein-27 kDa. ESTs (expressed sequence tags) encoding the predicted primary amino acid sequence of SRP-27 were then used to design PCR primers and to amplify a cDNA fragment from total rat mRNA which was used to, first, screen a mouse skeletal muscle cDNA library and, second, to produce a recombinant fusion protein to be used as an antigen to generate polyclonal antibodies against the C-terminal domain of SRP-27.
Predicted primary sequence of mouse SRP-27 and comparison with rat and human sequences
cDNA cloning, sequence analysis and membrane topology of SRP-27
We isolated the full length SRP-27 rat cDNA and compared the deduced amino acid sequence with the peptide sequence obtained from the protein sequence of the rabbit skeletal muscle junctional face membrane (Figure 1). The rat cDNA contains an open reading frame of 891 bp corresponding to a 297-amino-acid long polypeptide (Rattus norvegicus NCBI accession number EF690436). The deduced primary sequence of mouse skeletal muscle SPR-27 (Mus musculus NCBI accession number EF988666) predicts a protein of 298 amino acids with a molecular mass of 33 kDa. The predicted N-terminal sequences of both rat and mouse SPR-27 are 84% identical to the amino acid peptide sequence obtained from the 27 kDa protein of the rabbit junctional face membrane. Multiple sequence alignments reveal that the mouse skeletal muscle cDNA clone encodes a protein displaying 97% and 98% identity with rat and human SRP-27 (Figure 1). Transmembrane domain prediction (TMhMM) reveals four putative transmembrane segments.
Expression of the SRP-27/pEYFP fusion protein in COS-7 cells revealed two patterns of subcellular distribution: in approx. 40% of the transfected cells (437/1026), the fluorescence pattern was typically localized to the endoplasmic reticulum, as revealed by the reticular fluorescence originating around the nuclear membrane and extending into the cytoplasm. Interestingly, 57% (588/1026) of the transfected cells displayed large cisternae whose membranes are enriched in the YFP-tagged SRP-27 (Figures 2A and 2B). The exact nature of the stained cisternae is unknown; these structures may represent dilations of ER (endoplasmic reticulum) membrane induced by the over-expression of SRP-27.
SRP-27 is targeted to ER/SR membranes
In order to determine if native SRP-27 present in skeletal muscle is an intrinsic membrane protein, we extracted SR membranes with Na2CO3 at pH 11. Figure 2(C) shows that, after this treatment, SRP-27 is found in the insoluble membrane fraction, indicating that it is an integral protein of sarco(endo)plasmic reticulum membranes. Interestingly, we also noticed that SRP-27 tends to form homo-oligomers as indicated by the immunopositive band of approx. 55 kDa visible in Figure 2(C), lane 2. The immunospecificity of this band was verified by pre-adsorption of anti-SRP-27 with the GST-SRP-27 fusion protein. Under these conditions, both the upper and lower bands disappear (not shown).
The membrane topology of the C-terminal domain of the expressed SRP-27–YFP protein was established by mild trypsin proteolytic treatment of the total microsomal fraction isolated from transfected TSA cells. Western blot staining with polyclonal anti-GFP reveals that the 55 kDa recombinant SRP-27–EYFP fusion protein is degraded at increasing trypsin concentrations (Figure 2D, left). In control experiments carried out under similar conditions, the immunoreactivity of YFP was destroyed  (Figure 2D, right), whereas the reactivity of the intralumenal protein calreticulin was unaltered (Figure 2D, lower panel), indicating that trypsin was excluded from the lumenal space. Thus, since the C-terminal region of SRP-27 is degraded after trypsin digestion, it must be facing the cytoplasmic side of the endo(sarco)plasmic reticulum membrane.
Tissue distribution and developmental expression of SRP-27
The tissue distribution of SRP-27 was investigated by Western blot staining of proteins present in the microsomal fractions of different rat tissues using polyclonal anti-SRP-27. Figure 3(A) shows that the SRP-27 immunoreactive band is found in total rat microsomal membranes isolated from excitable tissues such as heart (lane 1), brain (lane 3) and skeletal muscle (lane 5) but is absent from lung, kidney, spleen, liver, stomach and intestine.
Expression of SRP-27 in different tissues and during development
We also determined the pattern of developmental expression of SRP-27 in skeletal muscles. Its expression appears to be dependent on both the developmental stage and on the muscle fibre type. Indeed, the content of SRP-27 in the total miscrosomal fraction of skeletal muscles increases at day 15 and peaks at approx. 1–2 months of post-natal development (Figures 3B and 3C). Furthermore, SRP-27 expression is 5- to 6-fold higher in fast twitch muscles compared to slow twitch muscles (Figures 3D and 3E). The increase in the SRP-27 content during post-natal development and its fibre type-specific expression suggest that this protein may be important in the post-natal maturation of skeletal muscle membranes. Since the SR is morphologically and functionally subspecialized, we analysed more precisely the subcellular distribution of SRP-27 within the SR membrane by a combination of biochemical and immunohistochemical techniques.
Subcellular distribution of SRP-27
Immunocytochemistry of muscle fibres from an adult mouse diaphragm (Figures 4A and 4B) indicates that SRP-27 is highly concentrated in the perinuclear ER region (arrows) and to a lesser extent is localized throughout the muscle fibres. The distribution pattern of SRP-27 between the myofibrils is consistent with its localization in the SR. However, direct comparison of its staining pattern with that of the RyR1 indicates that the sarcomeric SRP-27 overlaps, but does not precisely co-localize with, the RyR1. Double labelling of SRP-27, RyR1 or SERCA with α-actinin show that the band of the SRP-27-containing compartment is located over the I-band. There it partially overlaps with the RyR1-labelled triads and alternates with the dominant SERCA band corresponding to longitudinal SR at the A-band. Thus, SRP-27 appears to also localize in a separate subcompartment of the SR, which is adjacent to, but distinct from, that containing RyR or SERCA. Corroborating observations were made by double immunofluorescence labelling of SRP-27 with RyR in cultured mouse myotubes (supplementary Figure 2 at http://www.BiochemJ.org/bj/411/bj4110343add.htm). Whereas RyR1 was localized predominantly in clusters in the periphery of myotubes, SRP-27 was found in a membrane system throughout the myotubes. In addition, it accumulated between the nuclei in the centre of the myotubes and in the nuclear envelope, indicating that high concentrations of SRP-27 are also present in the ER.
Subcellular localization of SRP-27 in the ER and SR of skeletal muscle fibres
Western blot staining of SR fractions confirm data obtained by immunohistochemistry. SRP-27 is distributed in light (R2) and heavy (R4) SR membrane fractions, enriched in longitudinal SR membrane and in terminal cisternae (R4) respectively (Figure 4C). The presence of SRP-27 around the Z line and in the membrane fractions corresponding to longitudinal SR and terminal cisternae suggests that SRP-27 might be part of a macromolecular Ca2+ signalling complex involved in pumping Ca2+ back into the SR and/or in releasing it via the Ca2+ release channel to activate muscle contraction. To evaluate whether SRP-27 is involved in any aspect of SR Ca2+ handling, we analysed its distribution after separation of the SR membrane proteins by sucrose-density-gradient centrifugation. Terminal cisternae were solubilized in the presence of 1% CHAPS and 1 M NaCl and the solubilized proteins separated on a continuous sucrose gradient (5–20%). Western blot staining of the proteins from different fractions of the sucrose gradient with anti-SRP-27 (Figure 5, lower panel) shows that an important fraction of SRP-27 migrates in an intermediate density fraction containing other major SR proteins such as SERCA and calsequestrin (Figure 5, upper panel, Coomassie Brilliant Blue-stained gel). Interestingly, a fraction of the SRP-27 is also found in the sucrose-density-gradient fractions enriched in RyRs (asterisk). The distribution of SRP-27 in the density gradient was found to depend on the ionic strength of the sucrose gradient buffer. At low ionic strength (200 mM NaCl), the content of SRP-27 in the intermediate density fraction (enriched in SERCA and calsequestrin) is negligible and the bulk of SRP-27 is found in the fractions corresponding to a sucrose concentration of 15–18%, which also contain the RyR complex (not shown). The presence of SRP-27 in these heavy sucrose fractions could result from the aggregation of SRP-27, leading to the formation of large complexes that co-sediment with the RyR complex. However, we think that it is unlikely since aggregation of at least 100 SRP-27 molecules would be required to form a 2.5 MDa complex able to co-migrate with the RyR complex. On the other hand, the presence of SRP-27 in the fraction enriched in RyR after solubilization in 1% CHAPS at low ionic strength may suggest the existence of a macromolecular complex resulting from the direct or indirect association of SRP-27 with the RyR complex. In order to assess the existence of such an association, we performed two sets of experiments: in the first, solubilized SR proteins were pulled down using beads coated with maurocalcine, a specific effector of RyR. As shown in Figure 6(A), the RyR was found to interact with maurocalcine-coated beads. In addition, maurocalcine-coated beads also pulled-down SRP-27 (Figure 6A, right panel). Interestingly, SRP-27 obtained by in vitro translation was not pulled-down by maurocalcine-coated beads (Figure 6B), indicating that SRP-27 must bind to maurocalcine beads via another protein, most likely the RyR. In order to confirm that SRP-27 is part of a macromolecular complex containing RyRs, we performed co-immunoprecipitation experiments using anti-SRP-27 followed by Western blot analysis using anti-RyR, or co-immunoprecipitation experiments using anti-RyR followed by Western blot analysis using anti-SRP-27. As shown in Figure 6(C), these experiments clearly reveal the formation of a macromolecular complex formed by both SRP-27 and the RyR.
SRP-27 co-sediments with RyR1 on a sucrose gradient
SRP-27 is part of the RYR macromolecular complex
In conclusion, our results show that SRP-27 is widely distributed through muscle SR and ER, however, it also associates with the RyR, and thus is part of the SR macromolecular signalling complex.
While the present paper was being reviewed, Takeshima and co-workers published an article on the same protein .
The technical assistance of Malies Angebrand is gratefully acknowledged. This work was supported by grants from Telethon GGP05.g025, Ministero della Ricerca Scientifica e Tecnologica ex40% e 60%, Department of Anesthesia Basel University Hospital, HPRN-CT-2002–00331 from the European Union, and a grant from the Austrian Science Fund (P16532-B05).
- E–C coupling
expressed sequence tag
green fluorescent protein
sarcoplasmic/endoplasmic reticulum Ca2+-ATPase
sarcoplasmic reticulum protein of 27 kDa
yellow fluorescent protein